1. Field of the Invention
The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array. The invention also provides a process for producing an antigen or antigenic determinant in an ordered and repetitive array. The ordered and repetitive antigen or antigenic determinant is useful in the production of vaccines for the treatment of infectious diseases, the treatment of allergies and as a pharmaccine to prevent or cure cancer and to generate defined self-specific antibodies.
2. Related Art
Vaccine development for the prevention of infectious disease has had the greatest impact on human health of any medical invention. It is estimated that three million deaths per year are prevented worldwide by vaccination (Hillemann, Nature Medicine 4:507 (1998)). The most common vaccination strategy, the use of attenuated (i.e. less virulent) pathogens or closely related organisms, was first demonstrated by Edward Jenner in 1796, who vaccinated against smallpox by the administration of a less dangerous cowpox virus. Although a number of live attenuated viruses (e.g. measles, mumps, rubella, varicella, adenovirus, polio, influenza) and bacteria (e.g. bacille Calmette-Guerin (BCG) against tuberculosis) are successfully administered for vaccination, there is a risk for the development of serious complications related to a reversion to virulence and infection by the ‘vaccine’ organism, in particular in immunocompromised individuals.
The specific design of attenuated viruses is now enabled by recombinant DNA technology (i.e., genetic engineering) through the generation of deletion or mutation variants. For example, the administration of an engineered Simian Immunodeficiency Virus (SIV) with a deletion within the nef gene was shown to protect macaques from subsequent infection with a pathogenic SIV strain (Daniel et al., Science 258:1938–1941 (1992)). However, the progression of acquired immunodeficiency syndrome (AIDS)-like symptoms in animals administered attenuated SIV raises safety concerns (Baba et al., Science 267:1820–1825 ((1995)).
As an alternative approach, attenuated viruses or bacteria may be used as carriers for the antigen-encoding genes of a pathogen that is considered too unsafe to be administered in an attenuated form (e.g., Human Immunodeficiency Virus (HIV)). Upon delivery of the antigen-encoding gene to the host, the antigen is synthesized in situ. Vaccinia and related avipox viruses have been used as such carriers for various genes in preclinical and clinical studies for a variety of diseases (e.g., Shen et al., Science 252:440 (1991)). One disadvantage of this vaccination strategy is that it does not mimic the virion surface, because the recombinant protein is expressed on the surface of the host cell. Additionally, complications may develop in immunocompromised individuals, as evidenced by life-threatening disseminated vaccinia infections (Redfield, N. Eng. J. Med. 316:673 ((1998)).
A fourth vaccination approach involves the use of isolated components of a pathogen, either purified from the pathogen grown in vitro (e.g. influenza hemagglutinin or neuraminidase) or after heterologous expression of a single viral protein (e.g. hepatitis B surface antigen). For example, recombinant, mutated toxins (detoxified) are used for vaccination against diphtheria, tetanus, cholera and pertussis toxins (Levine et al., New generation vaccines, 2nd edn., Marcel Dekker, Inc., New York 1997), and recombinant proteins of HIV (gp120 and full-length gp160) were evaluated as a means to induce neutralizing antibodies against HIV with disappointing results (Connor et al., J. Virol. 72:1552 (1998)). Recently, promising results were obtained with soluble oligomeric gp160, that can induce CTL response and elicit antibodies with neutralizing activity against HIV-1 isolates (Van Cortt et al., J. Virol. 71:4319 (1997)). In addition, peptide vaccines may be used in which known B- or T-cell epitopes of an antigen are coupled to a carrier molecule designed to increase the immunogenicity of the epitope by stimulating T-cell help. However, one significant problem with this approach is that it provides a limited immune response to the protein as a whole. Moreover, vaccines have to be individually designed for different MHC haplotypes. The most serious concern for this type of vaccine is that protective antiviral antibodies recognize complex, three-dimensional structures that cannot be mimicked by peptides.
A more novel vaccination strategy is the use of DNA vaccines (Donnelly et al., Ann. Rev. Immunol. 15:617 (1997)), which may generate MHC Class I-restricted CTL responses (without the use of a live vector). This may provide broader protection against different strains of a virus by targeting epitopes from conserved internal proteins pertinent to many strains of the same virus. Since the antigen is produced with mammalian post-translational modification, conformation and oligomerization, it is more likely to be similar or identical to the wild-type protein produced by viral infection than recombinant or chemically modified proteins. However, this distinction may turn out to be a disadvantage for the application of bacterial antigens, since non-native post-translational modification may result in reduced immunogenicity. In addition, viral surface proteins are not highly organized in the absence of matrix proteins.
In addition to applications for the prevention of infectious disease, vaccine technology is now being utilized to address immune problems associated with allergies. In allergic individuals, antibodies of the IgE isotype are produced in an inappropriate humoral immune response towards particular antigens (allergens). The treatment of allergies by allergy immunotherapy requires weekly administration of successively increasing doses of the particular allergen over a period of up to 3–5 years. Presumably, ‘blocking’ IgG antibodies are generated that intercept allergens in nasal or respiratory secretions or in membranes before they react with IgE antibodies on mast cells. However, no constant relationship exists between IgG titers and symptom relief. Presently, this is an extremely time- and cost-consuming process, to be considered only for patients with severe symptoms over an extended period each year.
It is well established that the administration of purified proteins alone is usually not sufficient to elicit a strong immune response; isolated antigen generally must be given together with helper substances called adjuvants. Within these adjuvants, the administered antigen is protected against rapid degradation, and the adjuvant provides an extended release of a low level of antigen.
Unlike isolated proteins, viruses induce prompt and efficient immune responses in the absence of any adjuvants both with and without T-cell help (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235–270 (1997)). Although viruses often consist of few proteins, they are able to trigger much stronger immune responses than their isolated components. For B cell responses, it is known that one crucial factor for the immunogenicity of viruses is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasi-crystalline surface that displays a regular array of epitopes which efficiently crosslinks epitope-specific immunoglobulins on B cells (Bachmann & Zinkernagel, Immunol. Today 17:553–558 (1996)). This crosslinking of surface immunoglobulins on B cells is a strong activation signal that directly induces cell-cycle progression and the production of IgM antibodies. Further, such triggered B cells are able to activate T helper cells, which in turn induce a switch from IgM to IgG antibody production in B cells and the generation of long-lived B cell memory—the goal of any vaccination (Bachmann & Zinkemagel, Ann. Rev. Immunol. 15:235–270 (1997)). Viral structure is even linked to the generation of anti-antibodies in autoimmune disease and as a part of the natural response to pathogens (see Fehr, T., et al., J. Exp. Med. 185:1785–1792 (1997)). Thus, antigens on viral particles that are organized in an ordered and repetitive array are highly immunogenic since they can directly activate B cells.
In addition to strong B cell responses, viral particles are also able to induce the generation of a cytotoxic T cell response, another crucial arm of the immune system. These cytotoxic T cells are particularly important for the elimination of non-cytopathic viruses such as HIV or hepatitis B virus and for the eradication of tumors. Cytotoxic T cells do not recognize native antigens but rather recognize their degradation products in association with MHC class I molecules (Townsend & Bodmer, Ann. Rev. Immunol. 7:601–624 (1989)). Macrophages and dendritic cells are able to take up and process exogenous viral particles (but not their soluble, isolated components) and present the generated degradation product to cytotoxic T cells, leading to their activation and proliferation (Kovacsovics-Bankowski et al., Proc. Natl. Acad. Sci. USA 90:4942–4946 (1993); Bachmann et al., Eur. J. Immunol. 26:2595–2600 (1996)).
Viral particles as antigens exhibit two advantages over their isolated components: (1) Due to their highly repetitive surface structure, they are able to directly activate B cells, leading to high antibody titers and long-lasting B cell memory; and (2) Viral particles but not soluble proteins are able to induce a cytotoxic T cell response, even if the viruses are non-infectious and adjuvants are absent.
Several new vaccine strategies exploit the inherent immunogenicity of viruses. Some of these approaches focus on the particulate nature of the virus particle; for example see Harding, C. V. and Song, R., (J. Immunology 153:4925 (1994)), which discloses a vaccine consisting of latex beads and antigen; Kovacsovics-Bankowski, M., et al. (Proc. Natl. Acad. Sci. USA 90:4942–4946 (1993)), which discloses a vaccine consisting of iron oxide beads and antigen; U.S. Pat. No. 5,334,394 to Kossovsky, N., et al., which discloses core particles coated with antigen; U.S. Pat. No. 5,871,747, which discloses synthetic polymer particles carrying on the surface one or more proteins covalently bonded thereto; and a core particle with a non-covalently bound coating, which at least partially covers the surface of said core particle, and at least one biologically active agent in contact with said coated core particle (see, e.g., WO/94/15585).
However, a disadvantage of these viral mimicry systems is that they are not able to recreate the ordered presentation of antigen found on the viral surface. Antigens coupled to a surface in a random orientation are found to induce CTL response and no or only weak B-cell response. For an efficient vaccine, both arms of the immune system have to be strongly activated, as described above and in Bachmann & Zinkemagel, Ann. Rev. Immunol. 15:235 (1997).
In another example, recombinant viruses are being utilized for antigen delivery. Filamentous phage virus containing an antigen fused to a capsid protein has been found to be highly immunogenic (see Perham R. N., et al., FEMS Microbiol. Rev. 17:25–31 (1995); Willis et al., Gene 128:85–88 (1993); Minenkova et al., Gene 128:85–88 (1993)). However, this system is limited to very small peptides (5 or 6 amino acid residues) when the fusion protein is expressed at a high level (lannolo et al., J. Mol. Biol. 248:835–844 (1995)) or limited to the low level expression of larger proteins (de la Cruz et al., J. Biol. Chem. 263:4318–4322 (1988)). For small peptides, so far only the CTL response is observed and no or only weak B-cell response.
In yet another system, recombinant alphaviruses are proposed as a means of antigen delivery (see U.S. Pat. Nos. 5,766,602; 5,792,462; 5,739,026; 5;789,245 and 5,814,482). Problems with the recombinant virus systems described so far include a low density expression of the heterologous protein on the viral surface and/or the difficulty of successfully and repeatedly creating a new and different recombinant viruses for different applications.
In a further development, virus-like particles (VLPs) are being exploited in the area of vaccine production because of both their structural properties and their non-infectious nature. VLPs are supermolecular structures built in a symmetric manner from many protein molecules of one or more types. They lack the viral genome and, therefore, are noninfectious. VLPs can often be produced in large quantities by heterologous expression and can be easily be purified.
Examples of VLPs include the capsid proteins of hepatitis B virus (Ulrich, et al., Virus Res. 50:141–182 (1998)), measles virus (Wames, et al., Gene 160:173–178 (1995)), Sindbis virus, rotavirus (U.S. Pat. Nos. 5,071,651 and 5,374,426), foot-and-mouth-disease virus (Twomey, et al., Vaccine 13: 1603–1610, (1995)), Norwalk virus (Jiang, X., et al., Science 250:1580–1583 (1990); Matsui, S. M., et al., J. Clin. Invest. 87:1456–1461 (1991)), the retroviral GAG protein (PCT Patent Appl. No. WO 96/30523), the retrotransposon Ty protein p1, the surface protein of Hepatitis B virus (WO 92/11291) and human papilloma virus (WO 98/15631). In some instances, recombinant DNA technology may be utilized to fuse a heterologous protein to a VLP protein (Kratz, P. A., et al., Proc. Natl. Acad. Sci. USA 96: 19151920 (1999)).
Thus, there is a need in the art for the development of new and improved vaccines that promote a strong CTL and B-cell immune response as efficiently as natural pathogens.